Lateral flow immunoassay for complement activation and methods of use for point-of-care assessment of complement-associated disorders

ABSTRACT

A method for treating an individual at risk for a complement-associated disorder is provided, including: (a) obtaining a sample of a body fluid from the individual; (b) measuring a complement activation level in the sample via a point-of-care lateral flow immunoassay; (c) correlating the complement activation level in the sample to a risk of a complement-associated disorder by comparing the complement activation level in the sample to a reference level in a control, wherein a deviation in complement activation level in the sample compared to the reference level in the control indicates the individual is at risk for a complement-associated disorder; (d) selecting a treatment for the individual, based on the correlating of step (c); and (e) treating the individual with the treatment selected in accordance with step (d). Lateral flow immunoassays and a method of monitoring an individual suffering from a complement-associated disorder are also provided herein.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/409,297, filed Nov. 2, 2010, thecontents of which are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The presently disclosed subject matter relates to lateral flowimmunoassays for the measurement of complement activation. Specifically,the presently disclosed subject matter relates to a lateral flowimmunoassay for the quantitative measurement of intact C3 and iC3b in asample and methods of using the same in the evaluation and treatment ofindividuals at risk for complement-associated disorders such asinflammatory distress.

BACKGROUND

Inflammation is the physiological response of vascularized tissue toinjury, infection, and certain diseases. The inflammatory process is abiological requirement for wound healing after traumatic injury and forthe clearance of infection. However, inflammation can also damageself-tissue. For this reason, inflammation is often considered adouble-edged sword.

Complement is the most ancient arm of the immune system and is deeplyrooted in the inflammatory process. The complement protein cascade is afirst line of defense against invading microbes and a critical player inthe wound healing process. The complement cascade comprises more than 30serum and cellular proteins and plays important roles in innate andadaptive immunity. Complement activation can occur via three majorpathways: the classical, alternative, and lectin pathways. All threemajor pathways of complement activation converge on the central proteincomplement component 3 (C3). C3 is a central mediator of inflammationand is activated by most factors that cause inflammation. FIGS. 1 and 2provides schematic overviews of C3 and its activation products.

Complement, and C3 in particular, is associated with several diseaseindications, both acute and chronic. Examples include, but are notlimited to, trauma, respiratory distress, sepsis, other forms ofinfection, infectious diseases (e.g., hemorrhagic fevers), multipleorgan failure, age-related macular degeneration, rheumatoid arthritis,systemic lupus erythematosus, glomerular nephritis, ischemia/reperfusioninjury, inflammatory bowel disease, intracranial hemorrhage, myocardialinfarction, and cardiac arrest.

Severe trauma patients present unique clinical challenges. Accurateassessment of injury severity is important for initial intervention andpatient stabilization, as well as patient triage (e.g., following masscasualty incidents). Those patients admitted to the ICU, even afterinitial stabilization, remain at high risk for secondarylife-threatening complications involving organ dysfunction, respiratorydistress and sepsis, among others. Many of these conditions involvehyper-inflammatory events that can escalate rapidly and causesignificant damage before clinical symptoms are detected. These eventsare generally preceded by an increasingly unstable homeostasis of theinflammatory response. The ability to monitor inflammation frequently(e.g., every hour or two) and reliably at the earliest time pointsfollowing injury has tremendous clinical value and would improveclinical outcomes for critical care patients.

Several reports have shown that complement activation occurs immediatelyafter injury and correlates with severity of injury. In one study,circulating levels of complement protein in trauma patients were foundto correlate with patient outcome. See Hecke, et al., Circulatingcomplement proteins in multiple trauma patients—correlation with injuryseverity, development of sepsis, and outcome, Crit. Care Med. 25(12):2015-24 (1997). In this study, the authors measured the plasmaconcentrations of both C3a and total C3 directly after the injury and inthe ICU in the days following injury. They detected evidence ofcomplement C3 activation at the earliest time points following injury.However, complement activation was more pronounced in non-survivors thansurvivors for the first eight hours. At the earliest time points, thedegree of C3 activation correlated with patient outcome. Hecke et al.also found the ratio of the C3 split product, C3a, when taken as a ratioto total complement, was a better predictor of outcome than C3a alone.

A similar study by Zilow, et al., Complement activation and theprognostic value of C3a in patients at risk of adult respiratorydistress syndrome, Clin. Exp. Immunol. 79: 151-57 (1990),retrospectively found that monitoring of C3a and total C3 at frequent (6hr) intervals might be useful for identifying patients at high risk foror in the early stages of respiratory distress. These investigators drewthe first plasma sample within 2 hours of injury and repeated 6 hoursamplings for the first 48 hours and then at daily intervals thereafter.Zilow et al. found a significant correlation between C3a levels andC3a:total C3 ratio at 6 and 12 hours, as a well as from 5 days outward.

In the field of trauma care, the first hour after injury is sometimesreferred to as the “Golden Hour.” While not desiring to be bound bytheory, it is generally believed that intervention within the first hourafter traumatic injury greatly increases the outcome of the patient.Better diagnostic information provided earlier would help improve thecritical care specialist's intuition when making treatment decisions.

Heretofore, a point-of-care assay for measuring complement activationwithin the actionable window of treatment has not been known in the art.Although the associations between complement and disease or trauma havelong been recognized, C3 is monitored in only a small number of diseasesor conditions. Even in those instances, current assay methods havelimitations. First, in most cases, traditional complement assays aredirected to total C3 as the target analyte (for example, via turbidityassays and ELISA). Total C3 is a combination of intact (or native) C3and C3 activation and deactivation products. These tests generallydetect decreases in circulating C3 levels. Decreased levels of total C3therefore only measure C3 depletion due to massive activation. However,other factors such as diet or exercise can cause lower steady statelevels of C3. As total C3 assays do not measure turnover, the causes ofactivation cannot be distinguished. Furthermore, a test that measurestotal C3 cannot monitor the real-time changes in C3 activation signaturethat would be useful in directing patient care. For example, patientssuffering from trauma or systemic lupus (marked by decreased C3 levels)would benefit from improved C3 activation monitoring. Currently,treatment effectiveness for systemic lupus is measured by a return ofdepressed C3 levels to normal levels. However, the physician hasdifficulty in discerning whether the underlying disease process has beenhalted or just retarded sufficiently for homeostatic mechanisms toreturn C3 to physiologically normal levels.

A second limitation in current C3 testing is the time required toperform most assays. A typical ELISA assay for the detection ofcomplement activation requires hours to perform and the readyavailability of a laboratory and a skilled technician. This assayplatform is therefore not useful for indications of inflammatorydysfunction, in which biomarkers change on the order of minutes andclinical intervention is required on a similar timescale.

A third limitation in current C3 testing lies in the nature of theprotein cascade itself. Complement is notoriously fastidious and canbecome activated by virtue of standard analysis procedures (handling,storage, and exposure to foreign materials that contact C3 duringanalysis). Complement is very effective at lysing invading microbes andinitiating the wound healing response at sites of injury. Thiseffectiveness is due in part to the ability of C3 to be activated byforeign materials such as bacterial cell wall components. While thisproperty is useful in directing an immune response to new foreignpathogens, this same property presents formidable challenges toexperimental and diagnostic study. Materials such as plastics used insample handling, manipulation of the sample itself, and improper storageconditions can also trigger complement activation. The more processingand handling steps required to perform a given assay, the more falsepositives can be expected, due to activation of complement by virtue ofthe assay itself. These false positives complicate traditional testingand render current testing methods unsuitable for use in directingpatient care in near real-time.

A further consideration in complement activation testing is theselection of the best biomarker for detecting real time changes in theinflammatory response. C3 has several attractive qualities as abiomarker in inflammation. First, as the central protein of thecomplement system, C3 is activated by most stimuli that will causecomplement activation. Second, C3 activates in proportion to the degreeof injury or infection. Third, C3 responds in near real-time to aphysiological insult. Complement activation occurs in direct response toan agent causing crisis, in contrast to other acute phase inflammatorymarkers that take hours or days to respond. This rapid response propertyis not present in other biomarkers frequently used in the clinic.

Specifically, intact (or native) C3 is a valuable marker of inflammatorystatus. Intact C3 represents the amount of C3 available for activation.Total C3 represents intact C3 as well as all C3 activation products. Atpresent, standard complement assays generally measure total C3 viaturbidity assays or ELISA. Although technically easier to perform, totalC3 assays cannot detect C3 depletion as accurately as intact C3 assays.Monitoring intact C3, especially over time, is useful for followingmassive complement activation events, such as those that occur in traumaand other systemic complement activation indications. Monitoring intactC3 over time allows a clinician to detect the onset of animmunosuppressive state caused by depletion of C3. Further, intact C3may be more useful than total C3 when calculating complement activationindexes (e.g., the C3a:total C3 ratio used by Zilow and Hecke). IntactC3 assays have historically proven difficult to administer or dependupon, in part because intact C3 is very labile and can denature orself-activate if not handled properly.

The C3 split product, iC3b, is also a valuable marker of inflammatoryresponse. iC3b has a half-life of 30 to 90 minutes, serving as a lessvolatile (e.g., compared to C3a), but still rapidly responsivebiomarker. However, iC3b is present at much lower levels than intact C3in patient samples. Even a small degree of cross-talk (for example 1%)between intact C3 protein and the iC3b-specific assay produces a falsepositive iC3b signal at a level twice that of normal circulating iC3b.Hence, while a desirable marker of inflammation, heretofore iC3b hasposed significant challenges in diagnostic testing.

WO 2010/135717, by Zhang et al., published Nov. 25, 2010, is directed tomethods for assessing complement activation via the biomarkers intactC3, iC3b, and total C3. However, Zhang et al. is limited to traditionalsandwich-type immunoassays such as ELISA, requiring laboratoryprocessing and the expertise of skilled technicians. Further, the assaysand methods of Zhang et al. require sample preparation, storage, andhandling steps that are known to activate the labile intact C3 producefalse positive test results, impeding the ability to accurately measureintact C3. Moreover, the assays and methods of Zhang et al. requirehours to process and are thus incapable of providing the near real-timedata that can impact patient care in the earliest time points afterphysiological crisis.

The need persists for a rapid, point-of-care assay for the measurementof intact C3 and iC3b in a patient at risk for a complement-associateddisorder which minimizes complement activation due to sample handlingand allows a clinician to act upon changes in complement activationlevels in near real-time, in order to guide patient treatment andmonitor inflammatory response.

SUMMARY OF THE INVENTION

The present inventors have now developed a point-of-care method andassay for the qualitative and quantitative measurement of intact C3 andiC3b suitable for use in directing patient care at the earliest timepoints immediately following traumatic injury or other physiologicalcrises. By carefully selecting capturing and detecting antibody pairsthat avoid interfering cross-talk and applying the technology to alateral flow immunoassay platform, the inventors have surprisingly foundthat it is possible to detect and quantify the biomarkers intact C3 andiC3b, while avoiding the false positive results that have plagued moreconventional testing methods for these analytes.

Accordingly, a method for treating an individual at risk for acomplement-associated disorder is provided herein, the methodcomprising: (a) obtaining a sample of a body fluid from the individual;(b) measuring a complement activation level in the sample via apoint-of-care lateral flow immunoassay; (c) correlating the complementactivation level in the sample to a risk of a complement-associateddisorder by comparing the complement activation level in the sample to areference level in a control, wherein a deviation in complementactivation level in the sample compared to the reference level in thecontrol indicates the individual is at risk for a complement-associateddisorder; (d) selecting a treatment for the individual, based on thecorrelating of step (c); and (e) treating the individual with thetreatment selected in accordance with step (d).

In another embodiment, a lateral flow immunoassay for the point-of-caredetection of a marker of complement activation in a body fluid samplecomprising complement proteins is provided, the lateral flow immunoassaycomprising: a membrane strip; a detecting antibody that binds a firstepitope of the marker; a test line comprising a capturing antibody thatbinds a second epitope of the marker; and a control line comprising anantibody that binds a control analyte, wherein the marker is selectedfrom the group consisting of intact C3 and iC3b.

In still another embodiment, a lateral flow immunoassay for thepoint-of-care detection of markers of complement activation in a bodyfluid sample comprising complement proteins, the lateral flowimmunoassay comprising: a membrane strip; a first detecting antibodythat binds a first epitope of intact C3; a first test line comprising afirst capturing antibody that binds a second epitope of intact C3; asecond detecting antibody that binds a first epitope of iC3b; a secondtest line comprising a second capturing antibody that binds a secondepitope of iC3b; and at least one control line comprising an antibodythat binds a control analyte.

In another embodiment, a method for monitoring an individual who isreceiving treatment for a physiological condition and who is sufferingfrom a complement-associated disorder is provided, the methodcomprising: (a) obtaining serial samples of a body fluid from theindividual; (b) determining a complement activation level in each ofsaid samples via a point-of-care lateral flow immunoassay; (c) comparingthe complement activation levels in the serial samples to detect achange in a complement activation level over time; and (d) modifyingtreatment for the individual, based on the correlating of step (c).

These and other objects, features, embodiments, and advantages willbecome apparent to those of ordinary skill in the art from a reading ofthe following detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic overview of the complement system.Complement is activated by three major pathways, all of which convergeon the activation of intact C3. Proteolytic activation of C3 producesthe C3 split products C3a and C3b. C3b is further proteolyticallymodified to form iC3b, a biomarker for C3 activation. Intact C3 and iC3bare circled in the schematic.

FIG. 2 provides a schematic representation of C3 activation anddeactivation. Intact C3 is activated proteolytically to C3a and C3b.Some C3b molecules covalently attach to surfaces; others react withwater and stay in circulation. C3b is deactivated by the proteaseFactor 1. The first deactivation product is iC3b, which is formed by theactivity of Factor 1 removing a short peptide, C3f. iC3b is furtherdegraded to C3c and C3dg, the latter ultimately being degraded to C3d.

FIG. 3 is a schematic representation of specific recognition of intactC3 and iC3b by antibody pairs. (A) Intact C3 is recognized by twoantibodies: a first antibody recognizes C3a, which is only present inthe intact C3 molecule; a second antibody recognizes a region in C3dthat is present in both intact C3 and iC3b. The second antibodyparticipates in distinguishing C3 and its derivatives from other proteinmolecules but not intact C3 from iC3b. An alternate pair of antibodiesfor intact C3 include antibodies that recognized C3a and C3f. (B) TheiC3b protein is recognized by another antibody pair. The first antibodycontacts the protein at a neoepitope believed to be located near the C3gregion. This epitope is revealed once Factor I removes the C3f fragment.The neoepitope is occluded once Factor I degrades iC3b to C3c and C3dg.The second antibody recognizes the C3d epitope. An alternate pair foriC3b includes the aforementioned iC3b antibody and a second thatrecognizes activated C3d (Quidel® A250).

FIG. 4 is a schematic of one embodiment of a lateral flow immunoassay ofthe present invention.

FIG. 5 is a schematic representation of two embodiments of a lateralflow immunoassay. (A) shows a lateral flow immunoassay for detection ofa single analyte. (B) shows a lateral flow immunoassay for detection oftwo separate analytes (intact C3 and iC3b) in parallel membrane strips.

FIG. 6 is a depiction of three embodiments of single analyte lateralflow immunoassays. (A) shows a test cassette for a total C3 lateral flowimmunoassay. (B) shows a test cassette for an intact C3 lateral flowimmunoassay. (C) shows a test cassette for an iC3b lateral flowimmunoassay.

FIG. 7 is a depiction of two embodiments of a double analyte lateralflow immunoassay test cassette, for the assessment of intact C3 andiC3b. (A) shows a test cassette comprising two separate ports for sampleloading and two membrane strips in parallel, one for each analyte. (B)shows a test cassette with a single port for sample loading and twomembrane strips in parallel, one for each analyte.

FIG. 8 is a depiction of two embodiments of a triple analyte lateralflow immunoassay test cassette, for the assessment of total C3, intactC3, and iC3b. (A) shows a test cassette comprising three separate portsfor sample loading and three membrane strips in parallel, one for eachanalyte. (B) shows a test cassette with a single port for sample loadingand three membrane strips in parallel, one for each analyte.

FIG. 9 is a schematic representation of two embodiments of a lateralflow immunoassay. (A) shows a lateral flow immunoassay for detection ofa single analyte. (B) shows a lateral flow immunoassay for detection oftwo separate analytes (intact C3 and iC3b) in series on the samemembrane strip.

FIG. 10 is a depiction of two embodiments of a lateral flow immunoassayfor multiple analytes in series. (A) shows a lateral flow immunoassayfor detection of two analytes and a control in series on the samemembrane strip. (B) shows a lateral flow immunoassay for detection ofthree analytes and a control in series on the same membrane strip. Theanalytes are selected from total C3, intact C3, and iC3b for both (A)and (B).

FIG. 11 shows a comparison of sensitivities, dynamic range, test-to-testvariability, and assay time for three embodiments of the instant lateralflow immunoassays. (A) shows a standard curve graph of lateral flow fora test strip detecting iC3b without a cassette casing. (B) shows astandard curve graph of an embodiment of the lateral flow immunoassaywherein the test strips are enclosed in a cassette, which allows morecontrolled administration of the test sample volume. Concentration ofantibody solution used for gold conjugation is 0.5 mg/ml and BSA isincluded in the reaction mixture. (C) shows a standard curve graph ofanother embodiment of a test strip integrated into a cassette, whereinthe concentration of antibody solution used for gold conjugation is 1mg/ml and BSA is removed from the reaction mixture. Sensitivity of theassay reaches 10 ng/ml with a dynamic range extending to 10 ug/ml.

FIG. 12 shows sensitivity of a lateral flow immunoassay for iC3bdescribed herein. Sensitivity ranges from 10 ng/ml to 10 ug/ml. Standarderror is less than 3% at 20 minutes. R square=0.9892. Values wereverified by ELISA.

FIG. 13 shows sensitivity of a lateral flow immunoassay for intact C3described herein. Sensitivity ranges from 20 ng/ml to 10 ug/ml. Standarderror is less than 3% at 20 minutes. R square=0.9964. Values wereverified by ELISA. Error bars are shown, but are smaller than theplotted points.

FIG. 14 shows crosstalk between intact C3 and iC3b antibodies in lateralflow immunoassays.

FIG. 15 shows intact C3 and iC3b levels in basal tears from a singleindividual at 12 hour intervals, as assayed by lateral flow immunoassaysdescribed herein.

FIG. 16 shows intact C3 and iC3b levels in whole blood from a normalhealthy individual. Results show approximately 2500-fold more intact C3than iC3b in whole blood from a healthy donor.

FIG. 17 shows intact C3 and iC3b levels in whole blood from a healthyindividual. Results show approximately 333-fold more intact C3 than iC3bin whole blood from a healthy donor.

FIG. 18 shows intact C3 and iC3b levels in a normal individual 2 hoursafter heavy exertion (100 mile bicycle ride). Results show greater than1000-fold more intact C3 than iC3b in whole blood from a healthyindividual post-exertion.

FIG. 19 is a table of exemplary body fluids suitable for use with theassays and methods disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

“Analyte” means any entity, particularly a chemical, biochemical orbiological entity to be assessed, e.g., whose amount (e.g.,concentration or mass), activity, composition, or other property(ies)is/are to be detected, measured, quantified, evaluated, analyzed, etc.An “analyte” can be a single molecular species or can be composed ofmultiple distinct molecular species.

“Antibody” encompasses intact and/or full length immunoglobulins oftypes IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM, IgY,antigen-binding fragments or single chains of complete immunoglobulins(e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fdfragments, scFv (single-chain variable), and dAb fragments), and otherproteins that include at least one antigen-binding immunoglobulinvariable region, e.g., a protein that comprises an immunoglobulinvariable region, e.g., a heavy (H) chain variable region (VH) and alight (L) chain variable region (VL). The light chains of an antibodymay be of type kappa or lambda. An antibody may be polyclonal ormonoclonal. A polyclonal antibody contains immunoglobulin molecules thatdiffer in sequence of their complementarity determining regions (CDRs)and, therefore, typically recognize different epitopes of an antigen.Often a polyclonal antibody is derived from multiple different B celllines each producing an antibody with a different specificity. Apolyclonal antibody may be composed largely of several subpopulations ofantibodies, each of which is derived from an individual B cell line. Amonoclonal antibody is composed of individual immunoglobulin moleculesthat comprise CDRs with the same sequence, and, therefore, recognize thesame epitope (i.e., the antibody is monospecific). Often a monoclonalantibody is derived from a single B cell line or hybridoma. An antibodymay be a “humanized” antibody in which for example, a variable domain ofrodent origin is fused to a constant domain of human origin or in whichsome or all of the complementarity-determining region amino acids oftenalong with one or more framework amino acids are “grafted” from arodent, e.g., murine, antibody to a human antibody, thus retaining thespecificity of the rodent antibody.

In another embodiment, the capturing and/or detecting agents includeother ligands, such as natural receptors for activated C3 (e.g.,complement receptors 1, 2, and 3), aptamers, peptides, other smallmolecule ligands, and the like.

An aspect of certain embodiments of the invention is the selection ofantibodies for use as capture and detection agents. The inventorsdiscovered that many published and commercially available antibodiesexhibited crosstalk between intact C3 and various C3 cleavage productsor between different C3 cleavage products. For example, certainmonoclonal antibodies against human C3a show significant and unexpectedcross-reactivity with C3b and iC3b. It was recognized thatcross-reactivity could be a significant source of inaccuracy in certainsituations. Of particular concern in developing an assay for iC3b wascrosstalk between intact C3 and iC3b observed with many of the iC3bantibodies tested. Further testing showed that crosstalk between C3b andiC3b was even more significant with at least some of these antibodies.This was of concern because iC3b levels are expected to be present atmuch lower levels than intact C3 in patient samples. One aspect ofcertain embodiments of the invention is the selection of antibodies withspecificity for intact C3 or iC3b so as to minimize such crosstalk. Incertain embodiments, antibodies with specificity for intact C3 or iC3bare not substantially cross-reactive. In this context, “notsubstantially cross-reactive” means less than about 0.1% cross-reactive,meaning that a 1 ug/ml solution of C3 must register as less than about 1ng/ml of iC3b. The about 0.1% threshold is based on the physiologicallevels of intact C3 and iC3b in a normal individual. Normal iC3b levelsare approximately 0.5% that of total C3 in circulation. If C3 crosstalkcontributes more than about 25% to the iC3b signal in a complementactivation assay, the assay can produce false positive results thatabrogate the utility of the assay.

“Body fluid” means any fluid in the body that may be assayed forcomplement activation. Body fluids include, but are not limited to,whole blood, serum, plasma, urine, tears, saliva, wound exudate,broncheoalveolar lavage fluid, and cerebrospinal fluid. See FIG. 19 fora non-limiting list of suitable body fluids.

“Complement activation level” means the amount of complement (generallyC3) that is activated at a given time point. Amounts (i.e., levels) ofintact C3, iC3b, and/or total C3 are typically expressed in terms ofconcentration but may be expressed in terms of mass or weight.Concentration may be expressed in various ways, e.g., in terms ofmolarity, molality, mole fraction, mass fraction (mass of a substance ina mixture as a fraction of the mass of the entire mixture), mass perunit volume, etc. For purposes of description herein, concentration(e.g., mass per unit volume) will generally be used. Complementactivation level can also be described as a ratio of iC3b to intact ortotal C3, or as a ratio of C3a to total C3.

“Complement-associated disorder,” as used herein, refers to a disorderor condition characterized by a modification in complement activation.Examples of complement-associated disorders include, but are not limitedto, trauma, such as traumatic brain injury, spinal cord injury, surgery,and intracranial pressure; inflammatory distress, such as severeallergies, systemic inflammatory response syndrome (SIRS), multipleorgan failure (MOF), acute or adult respiratory distress syndrome(ARDS), septic shock, and shock; paroxysmal nocturnal hemoglobinuria(PNH); hereditary angiodema; renal disease, such as glomerularnephritis, infection, lupus nephritis, and renal disease requiring organtransplant; autoimmune disease, such as diabetes mellitus I,inflammatory bowel disease, Crohn's disease, multiple sclerosis,myasthenia gravis, rheumatoid arthritis, and systemic lupuserythematosus; ischemia/reperfusion injury; heart disease, such asmyocardial infarction and cardiac arrest; pregnancy, includingpreeclampsia and fetal hypoxia syndrome; ocular disease, such asage-related macular degeneration, dry eye syndrome, and ocularinfection; organ transplant, including transplant rejection, detectingimminent rejection, detecting infection, and monitoring adjustments inimmunosuppressive drug regimens; infection, including sepsis, pneumonia,bladder infection, urinary tract infection, and kidney infection; andneurological disorders, including multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, schizophrenia, and post-traumatic stressdisorder.

“Control” refers to a sample having a known reference level ofcomplement activation. In some embodiments, the control has a complementactivation level comparable to that of an individual who is notexperiencing a complement-associated disorder, such that a test samplehaving a complement activation level that is deviated compared to thecontrol is indicative of a complement-associated disorder. In certainembodiments, a complement-associated disorder is indicated when the testsample complement activation level is statistically significantlydeviated compared to the control.

“C3 activation signature,” as used herein, means changes in C3activation levels over time.

“Deviated” and “a deviation” as used herein, refer to statisticallysignificant deviations as compared to a reference level in a control.Depending on the analyte being assayed, a deviated test sample level maybe elevated or decreased relative to the control level.

“Decreased,” as compared to a reference level in a control, meansstatistically significantly decreased. In an acute inflammatoryresponse, intact C3 levels are depleted as C3 is broken down into itsactivation products. In certain embodiments, intact C3 levels areconsidered decreased as compared to a reference level in a control atabout 10%.

“Elevated,” as compared to a reference level in a control, meansstatistically significantly elevated. In an acute inflammatory response,iC3b levels increase as C3 is broken down into its activation products.In certain embodiments, a ratio of iC3b to intact C3 that is elevated ascompared to the normal ratio of 0.005 is indicative of C3 activation.

“Epitope” refers to the minimum portion of a molecule that is recognizedby, and thus determines the immunospecificity of, an antibody that bindsto such epitope. The term is also used herein to refer to the minimumportion of a molecule that is recognized by a non-antibody specificbinding agent. Unless otherwise indicated, it is assumed herein that aspecific binding agent that binds to a complement protein binds to anepitope present and accessible for binding in the native protein, i.e.,the epitope is not a neoepitope.

“Inflammatory distress” or “inflammatory dysfunction” occurs when theinflammatory response fails to resolve or remove the stimuli towardwhich the inflammatory response is directed. In such acute cases, theinflammatory response increases until homeostatic control over theprocess erodes. In one embodiment, a complement activation leveldetermined by the assays and methods disclosed herein correlatesdirectly with the severity of inflammatory distress being experienced byan individual. For example, when iC3b concentration is about 1-2.5% ofintact C3, the patient's inflammatory distress can be said to be mildlysevere. When iC3b concentration is about 2.5-5% of intact C3, thepatient's inflammatory distress can be said to be moderately severe.When iC3b concentration is over 5% of intact C3, the patient'sinflammatory distress is said to be highly severe. Understanding theseverity of a patient's inflammatory distress can inform a physician'streatment of the individual. For example, if the individual presentswith a highly severe inflammatory distress level, as indicated by theassays and methods disclosed herein, the physician can provide emergencymedical treatment within the earliest time points of inflammatorydistress, in order to minimize damage from inflammatory response.

“Label” refers to a moiety that facilitates the direct or indirectdetection and/or quantitative or relative measurement of a molecule towhich it is attached. A detectable label often produces a signal such asfluorescence, chemiluminescence, radioactivity, color, magnetic orparamagnetic properties, etc., that renders it detectable, e.g., by theuse of instruments that detect fluorescence, chemiluminescence,radioactivity, color, magnetic field, magnetic resonance, etc., or insome cases by visual inspection. The label may be, e.g., fluorescentsubstance; pigment; chemiluminescent or luminescent substance; coloredsubstance; magnetic substance; or a non-magnetic metal particle such asgold colloid. In a specific embodiment, the detecting antibodiessuitable for use in the instant methods and assays are conjugated to acolloidal gold label, which provides a color signal.

“Neoepitope” refers to an epitope that is generated or becomesdetectable as a result of proteolytic cleavage of a complement componentor cleavage product.

In certain embodiments of the assays and methods disclosed herein, thecomplement present in the body fluid sample tested is not substantiallyactivated by the assay or method itself. “Not substantially activated,”as used in this context, means that the lateral flow immunoassay resultsare substantially free of in vitro activation caused by the test methodsand/or materials. In this way, false positive test results forcomplement activation are avoided, since the lateral flow immunoassay israpid and requires less sample manipulation, thus avoiding many of thestimuli that contribute to in vitro complement activation.

“Point-of-care,” as used herein, refers to a device or method that canbe used or carried out at the bedside or site of injury of the patient.Point-of-care tests generally do not require shipping a sample to alaboratory for processing or the expertise of a skilled laboratorytechnician. The point-of-care methods and tests described herein allow aclinician to receive critical information at the patient's bedside, orat the site of traumatic injury or triage, which can direct patient careduring the critical first moments after a physiological crisis thattriggers complement activation.

“Reader” refers to an instrument suitable for the detecting of thesignal produced by the label. Various instruments are known in the artfor the detection of label signals in diagnostic testing. In a specificembodiment of the present invention, the label is colloidal gold and thereader is an instrument suitable for the qualitative and/or quantitativedetection of the color signal produced by the label. Suitable readersare available commercially from a variety of vendors, including BioAssayWorks (Ijamsville, Md.), the ESE-Quant from Qiagen (Hilden, Germany),Easterline LRE (Nordlingen, Germany), and Detekt Biomedical (Austin,Tex.). In a specific embodiment, the reader is a handheld reader thatquantifies the amount or concentration of intact C3, iC3b, or total C3.

“Treatment,” as used herein, encompasses any diagnostic, therapeutic,preventive, or remedial treatment administered to an individual. In someembodiments, treatment encompasses performing additional diagnostictesting on the individual. In other embodiments, treatment encompassestherapeutic treatment, such as a administering a therapeutic agent tothe individual. In certain embodiments, the therapeutic agent isselected from the group consisting of antibiotics, anti-inflammatoryagents, and inhibitors of complement. In other embodiments, treatmentencompasses modifying a treatment the individual has already received oris receiving. For example, in one embodiment treating an individual on aventilator encompasses optimizing the ventilator.

Overview of the Complement System

The complement system comprises more than 30 serum and cellular proteinsand plays important roles in innate and adaptive immunity. There arethree major pathways of complement activation. The classical pathway isprimarily activated by immune complexes, specifically IgG/IgM antibodiesbound to antigen. Other activators include lipopolysaccharide, myelin,polyanionic compounds, C-reactive protein (CRP), and microbial DNA andRNA. The lectin pathway is activated by polysaccharides withfree-mannose group and other sugars common to fungi and bacteria. Thealternative pathway is mediated by direct C3 activation by “foreign”substances that often include microbial cell wall components. All threemajor pathways of complement activation converge on the central proteincomplement component 3 (C3). C3 is a central mediator of inflammationand is activated by most factors that cause inflammation. See FIGS. 1and 2 for a schematic overview of the complement system.

The classical pathway is typically triggered by immune complexes, whichare complexes of antigen bound with antibodies, generally belonging tothe IgM or IgG isotypes. Immune complexes in turn bind to complementcomponent C1, which is comprised of C1q, C1r, and C1s. The binding ofC1q to an antibody-antigen complex triggers activation of C1r and C1s.Activated C1s then cleaves component C4 to produce C4a and C4b. C4b iscapable of covalent attachment to cell surfaces, although only aboutfive percent does so. The remaining 95 percent reacts with water to forma soluble, activated C4b. Component 2 can then associate with C4b, whichafter which it is activated by C1s to C2a and C2b. C4b and C2a combineto form C4bC2a, the classical pathway (CP) C3 convertase.

The CP convertase cleaves C3 to form C3a and C3b. Like activated C4b,C3b can covalently bind to cell surfaces or react with H₂O and stay insolution. Activated C3b has multiple roles. By itself, it can serve asan opsonin to make the decorated cell or particle more easily ingestedby phagocytes. In addition, C3b can associate with C4bC2a (the CP C3convertase) to for a C5 convertase. The complex, termed C4bC2aC3b istermed the CP C5 convertase. Alternatively, C3b can form the core ofanother C3 convertase called the alternative pathway (AP) C3 convertase.

The alternative pathway (AP) is another mechanism by which C3 can becomeactivated. It is typically activated by targets such as microbialsurfaces and various complex polysaccharides and other materials. Thisalternative pathway can also be initiated spontaneously by the cleavageof the thioester bond in C3 by a water molecule to form C3(H₂O). C3(H₂O)binds factor B, which allows factor D to cleave factor B to Ba and Bb.Bb remains associated with C3(H₂O) to form C3(H₂O)Bb complex, which actsas a C3 convertase and cleaves C3, resulting in C3a and C3b.

C3b formed either via this process or via the classical or lectinpathways binds to targets (e.g., on cell surfaces) and forms a complexwith factor B, which is subsequently cleaved by factor D and form Bb,resulting in C3bBb, which is termed the alternative pathway (AP) C3convertase. Binding of another molecule of C3b to the AP C3 convertaseproduces C3bBbC3b, which is the AP C5 convertase.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MB1-1 gene (known as LMAN-1 in humans) encodes a type 1 integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi. The MBL-2 gene encodes the solublemannose-binding protein found in serum. In the human lectin pathway,MASp-1 and MASP-2 are involved in proteolysis of C4 and C2, leading toC3 convertase, which lead to production of a C5 convertase as describedabove for the CP.

C5 convertase generated via any of the three pathways cleave C5 toproduce C5a and C5b. C5b then binds to C6, C7, and C8, which catalysespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The assembling MAC inserts itself into target cell membrane, forming apore delineated by a ring of C9 molecules. MAC formation causes celllysis of invading microbes, MAC formation on host cells can also causelysis, but not necessarily. Sublytic amounts of MAC on the membrane ofcells may affect cell function in a variety of way. The small cleavageproducts C3a, C4a, and C5a are anaphylatoxins and mediate multiplereactions in the acute inflammatory response. C3a and C5a are alsopotent chemotactic factors that attract immune system cells such asneutrophils and macrophages into the area of crisis.

Complement as a Biomarker for Physiological Crisis

Complement component C3 is useful as a general alert biomarker that thebody is responding to some form of physiological crisis, such as injury,infection, or other disease process. Complement has been associated witha wide variety of diseases, including lupus, arthritis, intracranialhemorrhage, diabetes, multiple sclerosis, heart disease, and age-relatedmacular degeneration. In many cases, the severity of disease correlateswith the level of complement activation. In some cases, complement canplay a role in disease pathology. In these cases, the body is not ableto successfully control the cause of inflammation, which goes from localto systemic. Complement activation can directly damage tissue or do soindirectly by over-activating cells and recruiting immune cells that inturn cause tissue destruction. Examples of over activation includeanaphylactic shock, multiple organ failure (MOF), acute respiratorydistress syndrome (ARDS), and systemic inflammatory response syndrome(SIRS).

Complement activation in the immediate and early post-trauma period hasbeen well documented and occurs by several different mechanisms, likelyinvolving all three major pathways. Release and activation ofproteolytic enzymes may directly activate complement components. Tissuedamage and disruption of the endothelial lining expose surfaces thatlack the endogenous complement inhibiting molecules that normallyprotect host tissues. These surfaces are susceptible to deposition ofC3b and alternative pathway activation. Complement activation is alsotriggered by reperfusion of tissues following post-traumatic ischemia.

Multiple lines of evidence suggest that complement activation is animportant factor in many of the complications of severe trauma,contributing significantly to FR injury, ARDS, MODS, secondary CNSinjury, and sepsis. First, it is clear that complement activation is acommon occurrence in the immediate post-trauma period in human traumavictims, and several studies have provided evidence suggesting that theextent of complement activation correlates positively with pooroutcomes. Second, there is considerable evidence that complementactivation is a major cause of FR injury in animal models of trauma aswell as in human trauma victims. Third, numerous studies havedemonstrated that complement deficiency or administration of complementinhibitors reduces tissue damage and improves outcomes in a variety ofexperimental models including hemorrhage, FR injury, and CNS injury.

Several studies measured complement activation in trauma patients atsequential time points following severe trauma and investigated theexistence of a correlation between complement activation and injuryseverity. Adverse outcomes such as ARDS, multi-organ failure, sepsis,and death were also monitored in relation to complement activation. Inone study, complement parameters were determined over 14 days in traumapatients at risk of ARDS. All patients showed a decrease in serum levelsof C3, C4, C5 and of the inhibitors C1-INH, complement factor H (CFH),and complement factor I (CFI) in the first 24 hours, indicatingconsumption by high levels of complement activation. See Catania et al.,Immunological consequences of trauma and shock, Ann. Acad. Med.Singapore 28:120-32 (1999); Hecke, et al., Circulating complementproteins in multiple trauma patients—correlation with injury severity,development of sepsis, and outcome, Crit. Care Med. 25(12): 2015-24(1997); Huber-Lang et al., Complement-induced impairment of innateimmunity during sepsis, J. Immunol. 169:3223-31 (2002); Kang et al.,Change of complement system predicts the outcome of patients with severethermal injury, J. Burn Care Rehabil. 24:148-53 (2003); and Younger etal., Detrimental effects of complement activation in hemorrhagic shock,J. Appl. Physiol. 90:441-46 (2001).

Lateral Flow Assays for the Detection and Quantification of ComplementActivation and their Methods of Use

The presently disclosed assays and methods provide several advantagesover previous complement assays and methods known in the art: First, theinstant assays and methods are suitable for point-of-care use, producingresults in a matter of minutes, rather than hours. The rapid return ofresults allows a clinician to act upon changes in C3 activation in nearreal-time to direct patient care during the critical first moments aftertraumatic injury or at the onset of physiologic crisis. The assays andmethods are relatively easy to use and do not require the availabilityof an outside laboratory or a skilled lab technician. Second, theinstant assays and methods require fewer handling steps, and thusminimize intact C3 activation due to handling and processing, whichleads to false positive test results. Third, the assays and methodsdescribed herein employ antibody pairs carefully selected to allow formeasurement of the complement proteins intact C3 and/or iC3b, C3's majoractivation biomarker. This more precise measurement of complementactivation, in comparison to traditional assays of total C3, permitsanalysis of turnover and actual amount of C3 remaining and available foractivation.

In one embodiment, a method for treating an individual at risk for acomplement-associated disorder is provided, the method comprising: (a)obtaining a sample of a body fluid from the individual; (b) measuring acomplement activation level in the sample via a point-of-care lateralflow immunoassay; (c) correlating the complement activation level in thesample to a risk of a complement-associated disorder by comparing thecomplement activation level in the sample to a reference level in acontrol, wherein a deviation in complement activation level in thesample compared to the reference level in the control indicates theindividual is at risk for a complement-associated disorder; (d)selecting a treatment for the individual, based on the correlating ofstep (c); and (e) treating the individual with the treatment selected inaccordance with step (d).

In another embodiment of the method, the complement-associated disorderis selected from the group consisting of trauma, inflammatory distress,autoimmune disorders, intracranial hemorrhage, infection, transplantrejection, ocular disease, heart disease, ischemia/reperfusion injury,age-related macular degeneration, paroxysmal noctural hemoglobinuria(PNH), hereditary angiodema, renal disease, pregnancy-associateddisorders, and neurological disorders. In a specific embodiment, thecomplement-associated disorder is inflammatory distress. Inflammatorydistress, also known as inflammatory dysfunction, includes a variety ofdiseases and conditions associated with hyperinflammation. Examples ofdiseases and conditions associated with inflammatory distress include,but are not limited to, organ failure, systemic inflammatory responsesyndrome (SIRS), adult respiratory distress syndrome (ARDS), sepsis, andpneumonia.

In one embodiment of the method, the body fluid obtained from theindividual is selected from the group consisting of whole blood, serum,plasma, urine, tears, saliva, wound exudate, broncheoalveolar lavagefluid, and cerebrospinal fluid. See FIG. 19 for a non-limiting list ofsuitable body fluids. In a specific embodiment, the body fluid isobtained from the individual within one hour of a physiological eventtriggering complement activation. In another specific embodiment, thebody fluid is whole blood.

In one embodiment of the method, the lateral flow immunoassay detectsthe presence or absence of one or more of intact C3 and iC3b in thesample. In another embodiment of the method, the lateral flow assaydetects the presence of total C3. In another embodiment, the lateralflow immunoassay is read by a reader. In a more specific embodiment, thereader quantifies a concentration of one or more of intact C3 and iC3bin the sample. In another specific embodiment, the reader quantifies aconcentration of total C3 in the sample.

Complement activation levels are assessed for deviation from a referencevalue of a control which indicates complement is activated in theindividual. In certain embodiments, the level or concentration of iC3bin the test sample is elevated in comparison to a control, indicating C3is activated and has been further split into its activation product,iC3b. In other embodiments, the level or concentration of intact C3 isdecreased in comparison to a control, indicating intact C3 has beenconverted to its breakdown or activation products and is hence depletedin the individual.

Complement activation level correlates to a severity of inflammatorydistress: the higher the complement activation level, the greater therisk of developing inflammatory distress and/or the greater the severityof inflammatory distress experienced by the individual. Therefore, inanother embodiment of the method, the complement-associated disorder isinflammatory distress and the concentration of one or more of intact C3and iC3b correlates to a severity of inflammatory distress.

The complement activation level determined by the instant methodprovides point-of-care diagnostic information that can direct patientcare. Based on the risk of complement-associated disorder correlated instep (c), the clinician can select the appropriate treatment for theindividual. In one embodiment, the treatment comprises performingadditional testing on the individual to determine the cause ofinflammatory distress. For example, severe trauma patients that requireventilator assistance for breathing are at risk for acute respiratorydistress caused either by Ventilator Associated Pneumonia (VAP) ornon-infectious inflammatory dysfunction. A level of complementactivation may indicate active or imminent inflammatory dysfunctionbefore clinical signs of respiratory crisis are presented. The instantassays and methods may indicate whether the individual is experiencingVAP or non-infectious respiratory distress. Alternatively, the instantassays and methods may indicate additional testing (such asbroncheoalveolar lavage (BAL)) at a time point earlier than is nowstandard practice If the individual is suffering from VAP, the treatmentmay comprise administering a therapeutic agent such as an antibiotic orset of antibiotics. If the inflammatory dysfunction is caused bynon-infectious means, a therapy may be selected from the groupconsisting of ventilator adjustment, anti-inflammatory agents, andinhibitors of complement.

If the individual is suffering from traumatic brain injury orintracranial hemorrhage, the additional testing may comprise obtaining acerebrospinal fluid sample for additional analysis. If the individual issuffering from a wound, including a non-healing wound, the furthertesting may comprise obtaining a sample of wound exudate for additionalanalysis.

Many inhibitors of complement are known in the art and suitable for usein the instant methods. In one embodiment, the inhibitor of complementis selected from the group consisting of natural complement inhibitorsand derivatives thereof, compstatin and analogs thereof, anti-membraneattack complex (MAC) antibodies, anti-C3 antibodies, anti-05 antibodies,C3a receptor antagonists, and C5a receptor antagonists. Examples ofadditional complement inhibitors can be found, for example, in Emlen etal., Therapeutic complement inhibition: new developments, Semin. Thromb.Hemost. 36(6):660-68 (2101); Wagner et al., Therapeutic potential ofcomplement modulation, Nat. Rev. Drug Discov. 9(1):43-56 (21010); andRicklin et al., Complement-targeted therapeutics, Nat. Biotechnol.25(11):1265-75 (2007), the contents of which are incorporated byreference herein in their entirety.

One of the benefits of the instant method is the rapid return ofresults, which enables a clinician to direct patient care in response tochanges in complement activation in near real-time. Whereas previousassays for complement activation known in the art require fulllaboratories, skilled technicians, and hours to complete, the instantmethods and assays provide results in a much shorter time frame. In oneembodiment, the instant method provides a measurement of the complementactivation level in the sample in about 30 minutes or less. In a morespecific embodiment, the method provides a complement activation levelin the sample in about 30, about 25, about 20, about 15, about 10, about5, or about 3 minutes or less. The rapidity of the method enables theclinician to determine a complement activation level and select anappropriate therapy in response, during a clinically-meaningful timeperiod. Indeed, the instant methods can be carried out at the bedside oreven at the site of traumatic injury—for example, in an ambulance or intriaging a patient on the battlefield—and the complement activationlevel determined by the assay and method can direct patient care withinthe critical first hour post-trauma.

In another aspect of the invention, a method is provided for monitoringan individual who has received or is receiving treatment for aphysiological condition and who is known to be suffering from acomplement-associated disorder, the method comprising: (a) obtainingserial samples of a body fluid from the individual; (b) determining acomplement activation level in each of said samples via a point-of-carelateral flow immunoassay; (c) comparing the complement activation levelsin the serial samples to detect a change in a complement activationlevel over time; and (d) modifying treatment for the individual, basedon the correlating of step (c). Serial blood samples are collected andtested to monitor complement activation levels in response to thetreatment.

In one embodiment, the complement-associated disorder is selected fromthe group consisting of systemic lupus erythematosus, inflammatorydistress, autoimmune disorders, intracranial hemorrhage, bacteremia,transplant rejection, ocular disease, heart disease,ischemia/reperfusion injury, age-related macular degeneration,paroxysmal noctural hemoglobinuria (PNH), hereditary angiodema, renaldisease, pregnancy-associated disorders, and neurological disorders, andtrauma including patients at risk for inflammatory dysfunction such asventilator associated pneumonia (VAP), respiratory distress, andmultiple organ failure.

As with other methods disclosed herein, in certain embodiments, the bodyfluid is selected from the group consisting of whole blood, serum,plasma, urine, tears, saliva, wound exudate, broncheolar lavage fluid,and cerebrospinal fluid. In one embodiment, the lateral flow immunoassaydetects the presence or absence of one or more of intact C3 and iC3b inthe sample. In another embodiment, the lateral flow immunoassay is readby a reader which is capable of quantifying a concentration of intactC3, iC3b, or total C3.

In certain embodiments, the clinician will detect a decrease incomplement activation in response to the treatment the patient isreceiving. Accordingly, the clinician will modify the individual'streatment by adjusting the dosing of medications administered, such asanti-inflammatory agents or complement inhibitors, or discontinuingtreatment once complement levels have returned to normal (i.e., a levelin an individual who is not experiencing a complement-associateddisorder). In other embodiments, the clinician will detect a rise incomplement activation levels in response to the treatment the patient isreceiving. Accordingly, the clinician will modify the individual'streatment by increasing the dosage of medications, such asanti-inflammatory agents or complement inhibitors, until a desiredstabilization or decrease in complement activation levels is achieved.If no change in complement activation level is detected, the clinicianmay modify the individual's treatment or may elect to maintain theindividual's treatment regimen until a change in complement activationlevels is observed.

Referring to FIG. 4, the lateral flow immunoassay described herein iscomprised of a cellulose membrane strip 3, upon which is disposed asample pad 1 to absorb the sample fluid and allow gradual migration ofthe sample-and-particle-conjugate immune complexes, a wick 6 at thedistal end of the strip that absorbs the liquid sample and conjugatematerial to facilitate capillary migration through the cellulosemembrane strip 3, and a particle conjugate pad 2 comprising a detectingantibody bound to a label, or detection conjugate. The cellulosemembrane strip 3 is the test zone region, upon which is disposed a testline 4, comprising monoclonal or polyclonal antibodies striped forcapturing the detection conjugate and a control line 5, comprising anantibody that binds a control analyte, such as IgG, and indicates to theuser that the test was successfully run. The lateral flow immunoassayfurther comprises a polyester film backing 7 attached to the cellulosemembrane strip 3, and a pressure-sensitive laminate film backing 8. Eachlateral flow immunoassay is packaged in a Mylar® zero-vapor barrierpouch.

When a test sample is applied to the sample pad 1, the sample migratesfrom the sample pad 1 through the particle conjugate pad 2, where anytarget analyte present will bind to the detecting antibody conjugate.The sample then continues to migrate across the membrane 3 until itreaches the test line 4 where the target/conjugate complex will bind tothe immobilized antibodies producing a visible line on the membrane. Thesample then migrates further along the membrane strip 3 until it reachesthe control line 5, where excess antibody conjugate that did not bindthe test line will bind the control line and produce a second visibleline on the membrane. The control line ligand is often an antibodyagainst the Fc region of the conjugated antibody. This control lineindicates that the sample has migrated across the membrane as intended.

In certain embodiments, the lateral flow immunoassay comprises a singlemembrane strip for the detection of a single analyte. In otherembodiments, the lateral flow immunoassay detects two or more analytes.When the lateral flow immunoassay detects two or more analytes, the testcan be configured with multiple membrane strips arranged in parallel(see, for example, the schematic of FIG. 5(B)), or with multiple testlines arranged in series on a single membrane strip (see, for example,the schematic of FIG. 9(B)).

Referring to FIG. 6, in some embodiments, the lateral flow immunoassaymembrane strip is enclosed in a test, cassette 9 having a port 10 forinstilling the test sample and a window 11 for viewing the test results.The lateral flow immunoassays of FIG. 6 are configured to assay for asingle analyte and each comprise one test line 4 and one control line 5.

Referring to FIG. 7, in some embodiments, the lateral flow immunoassayis configured to test for two analytes in a single test cassette inparallel. In some embodiments, the lateral flow immunoassay comprisestwo ports 10 for instilling the test samples and a separate membranestrip 3 for each analyte (see FIG. 7(A)). In other embodiments, thelateral flow immunoassay comprises one port 10 for instilling the sampleand a separate membrane strip 3 for each analyte (see FIG. 7(B)).

Referring to FIG. 8, in some embodiments, the lateral flow immunoassayis configured to test for three analytes in a single test cassette inparallel. In some embodiments, the lateral flow immunoassay comprisesthree ports 10 for instilling the test sample and a separate membranestrip 3 for each analyte (see FIG. 8(A)). In other embodiments, thelateral flow immunoassay comprises one port 10 for instilling the testsample and a separate membrane strip 3 for each analyte (see FIG. 8(B)).

Referring to FIG. 10, in certain embodiments, the lateral flowimmunoassay is configured to test for multiple analytes in a single testcassette in series. FIG. 10(A) depicts a test cassette comprising amembrane strip 3 with two test lines 4 and one control line 5 arrangedin series. FIG. 10(B) depicts a test cassette comprising a membranestrip 3 with three test lines 4 and one control line 5 arranged inseries.

The lateral flow immunoassay presently disclosed provides qualitativeand/or quantitative detection of the target markers. Qualitatively, twoclear lines on the membrane is a positive result, whereas a single linein the control zone is a negative result.

In one embodiment, a lateral flow immunoassay for the point-of-caredetection of a marker of complement activation in a body fluid samplecomprising complement proteins is provided, the lateral flow immunoassaycomprising: a membrane strip; a detecting antibody that binds a firstepitope of the marker; a test line comprising a capturing antibody thatbinds a second epitope of the marker; and a control line comprising anantibody that binds a control analyte, wherein the marker is selectedfrom the group consisting of intact C3 and iC3b.

In one embodiment, the detecting antibody comprises a label thatprovides a signal that can be read visually by a clinician orelectronically via a commercial reader. Various labels are suitable foruse in the instantly disclosed assays. In a specific embodiment, thelabel is colloidal gold.

Detecting and capturing antibody pairs must be carefully selected toavoid interfering crosstalk between C3 and iC3b. The primary concern isintact C3 producing a signal in an assay for the detection of iC3b. Asboth molecules are derived from the same protein molecule, crosstalk canpresent a problem. As C3 is present at levels about 200 times higherthan iC3b in normal individuals, even a slight degree of crosstalk canhave a major impact on the accurate measurement of iC3b and C3activation. This is further complicated by the fact that improperhandling, improper storage, and even reagents themselves can cause invitro C3 activation. Surprisingly, Applicants discovered that not allantibodies suitable for use in traditional ELISA assays are equallysuitable for use in the assays of the instant invention. Tables 1 and 2below show the difficulties in identifying antibody pairs suitable foruse in the assays of the instant invention. The inventors analyzed 19pairs of antibodies in the intact C3 immunoassay and 18 pairs ofantibodies in the iC3b lateral flow immunoassay. Of these pairs, Hycult®HM2075 and MP Biomedicals® 55237 yielded the best results, with nocross-reactivity, in the intact C3 lateral flow immunoassay. Quidel®A209 with either MP Biomedicals® 55237 or Quidel® A250 yielded the bestresults in the iC3b lateral flow immunoassay. Interestingly, theinventors noted that antibody pairs suitable for use in traditionalELISA assays are not necessarily equally suitable for use in the lateralflow immunoassays described herein. For example, Hycult® HM2198 yieldedan assay with about a 1% cross-reactivity, with considerabletest-to-test variability. This cross-reactivity produced a falsepositive iC3b signal at a level of twice that of normal circulatingiC3b. As actual double or tripling of iC3b levels would be signs ofmassive complement activation, a lateral flow immunoassay with 1%cross-reactivity is without clinical utility. MP Biomedicals® (55237)worked far better, producing cross-reactivity of less than about 0.5%(about 0.05%), compatible with clinical utility. However, it isnoteworthy that both antibodies performed equally well in traditionalELISA assays.

TABLE 1 Antibody Screening Results in intact C3 assay capturing antibodydetection antibody species antigen supplier species antigen suppliernotes mouse C3a Hycult (HM2075) goat C3 MP Biomedicals (55237) no crossreactivity under assay conditions mouse C3a Quidel (A203) goat C3 MPBiomedicals (55237) assay to assay variance too high chicken C3a GenTex(GTX78198) rabbit C3d Abcam (ab15981) no positive readings (doesn'twork) mouse C3a Quidel (A203) rabbit C3d Abcam (ab15981) cross-reactswith C3b/iC3b +++ goat C3a SantaCruz (sc17237) rabbit C3d Abcam(ab15981) the Ab may only react with C3a, not intact C3 mouse C3a Hycult(HM2073) rabbit C3d Abcam (ab15981) cross reacts with C3b/iC3b + mouseC3a Hycult (HM2074) rabbit C3d Abcam (ab15981) no positive readingschicken C3a Abcam (ab48580) rabbit C3d Abcam (ab15981) no positivereadings mouse C3a Hycult (HM2073) chicken C3a Abcam (ab48580) nopositive readings mouse C3a Quidel (A203) chicken C3a Abcam (ab48580) nopositive readings chicken C3a Abcam (ab48580) goat C3 MP Biomedicals(55237) cross reacts with C3b/iC3b +++ mouse C3a Hycult (HM2073) goat C3MP Biomedicals (55237) cross reacts with C3b/iC3b +++ goat C3a SantaCruz(sc17237) goat C3 MP Biomedicals (55237) similar to HM2073, better indiluted serum rabbit C3d Abcam (ab15981) mouse C3a Quidel (A203) whenanti-C3d is capture Ab, it binds to C3b and rabbit C3d Abcam (ab15981)chicken C3a GenTex (GTX78198) iC3b as well, which prevents efficientbinding of rabbit C3d Abcam (ab15981) goat C3a SantaCruz (sc17237)intact C3 when analying mixed samples. rabbit C3d Abcam (ab15981)chicken C3a Abcam (ab48580) rabbit C3d Abcam (ab15981) mouse C3a Hycult(HM2073) rabbit C3d Abcam (ab15981) mouse C3a Hycult (HM2074)

TABLE 2 Antibody Screening Results in iC3b assay capturing antibodydetection antibody species antigen supplier species antigen suppliernotes mouse iC3b Quidel (A209) goat C3 MP Biomedicals (55237) no crossreactivity under assay conditions, good signal mouse iC3b AbD serotec(MCA2607) goat C3 MP Biomedicals (55237) cross reacts with C3b/C3c athigh conc. mouse iC3b AbD serotec (MCA2607) rabbit C3d Abcam (ab15981)lower signal strength than using anti-C3 mouse iC3b Quidel(A209) rabbitC3d Abcam (ab15981) lower signal strength than using anti-C3 mouse iC3bQuidel(A209) rat C3d Hycult (HM2198) good signal, lower than usinganti-C3 mouse iC3b Quidel(A209) rat C3g Hycult (HM 2199) no signal mouseiC3b Quidel(A209) mouse neo C3d Quidel (A250) lower signal strength thanusing HRP-anti C3, better specificity than using anti-C3d rat iC3bHycult (HM2199) goat C3 MP Biomedicals (55237) good signal rat iC3bHycult (HM2199) mouse active C3 Hycult (HM2168) weak signal rat iC3bHycult (HM2199) mouse active C3 Hycult (HM2257) no signal rat iC3bHycult (HM2199) mouse iC3b Quidel (A209) no signal rat iC3b Hycult(HM2199) mouse neo C3d Quidel (A250) no signal mouse active C3 Hycult(HM2168) goat C3 MP Biomedicals (55237) too much crosstalk with C3 mouseactive C3 Hycult (HM2168) rat C3g Hycult (HM 2199) weak signal mouseactive C3 Hycult (HM2257) goat C3 MP Biomedicals (55237) no signal mouseactive C3 Hycult (HM2257) rat C3g Hycult (HM 2199) no signal mouse C3alpha Meridian (H54189M) goat C3 MP Biomedicals (55237) no signal mouseneo C3d Quidel (A250) rat C3g Hycult (HM 2199) very low signal

In one embodiment, the marker is intact C3 and the detecting antibodybinds a first epitope of intact C3, wherein the first epitope is a C3adomain which is present on intact C3 and which is lost upon activationof C3. In a further embodiment, the marker is intact C3 and thecapturing antibody binds a second epitope on C3, wherein the secondepitope is a region in the C3d domain which is present on intact C3,C3b, iC3b, and C3d. See FIG. 3(A).

In another embodiment, the marker is iC3b and the detecting antibodybinds a first epitope of iC3b, wherein the first epitope is a neoepitopeon iC3b which is revealed when C3b is deactivated to iC3b and which isoccluded when iC3b is further degraded to C3c and C3d. In a furtherembodiment, the marker is iC3b and the capturing antibody binds a secondepitope on iC3b, wherein the second epitope is a neoepitope present onlyon C3b, iC3b, and C3dg. See FIG. 3(B).

In a very specific example, the marker is intact C3, the capturingantibody is Hycult® HM2075 and the detecting antibody is MP Biomedicals®55237. In another very specific example, the marker is iC3b, thecapturing antibody is Quidel® A209 and the detecting antibody is MPBiomedicals® 55237. In another very specific example, the marker isiC3b, the capturing antibody is Quidel® A209 and the detecting antibodyis Quidel® A250.

Various body fluids are suitable for use in the lateral flowimmunoassays and methods of the instant invention. See, for example,FIG. 19, for a non-limiting list of suitable body fluids for use in theassays and methods described herein. In one embodiment, the body fluidis selected from the group consisting of whole blood, serum, plasma,urine, tears, saliva, wound exudate, bronchoalveolar lavage fluid, andcerebrospinal fluid. In a specific embodiment, the body fluid is wholeblood.

One skilled in the art will appreciate that various control analytes aresuitable for use in the lateral flow immunoassays of the instantinvention to provide verification that the assay was successfullycompleted. In one embodiment, the control analyte is IgG.

Another advantage of the instant lateral flow immunoassay is theavoidance of substantial complement activation in the sample by virtueof the test itself, which can lead to false positive results. It is wellknown that C3 is a fastidious protein capable of self activation due tosample handling, storage, and contact with foreign materials orsubstances. Thus, the nature of C3 can lead to false positives intraditional ELISA and turbidity assays for complement activation thatinvolve extensive sample handling and multiple steps. The instantlateral flow immunoassay avoids such false positives by reducing and/oreliminating sample preparation and handling steps. Accordingly, in oneembodiment of the instant lateral flow assay, complement in the bodyfluid sample is not substantially activated experimentally by thelateral flow immunoassay.

In an alternative embodiment, it is desirable to have a lateral flowimmunoassay that can detect more than one marker of complementactivation in a single assay. For example, a dual lateral flowimmunoassay that can qualitatively and quantitatively detect both intactC3 and iC3b in the same aliquot of a body fluid is highly desirable.Hence, in another embodiment, a lateral flow immunoassay for thepoint-of-care detection of markers of complement activation in a bodyfluid sample comprising complement proteins is provided, the lateralflow immunoassay comprising: a membrane strip; a first detectingantibody that binds a first epitope of intact C3; a first test linecomprising a first capturing antibody that binds a second epitope of theintact C3; a second detecting antibody that binds a first epitope ofiC3b; a second test line comprising a second capturing antibody thatbinds a second epitope of iC3b; and at least one control line comprisingan antibody that binds a control analyte.

In one embodiment, the first and second detecting antibodies comprise alabel that provides a signal. Various labels are suitable for use in theinstantly disclosed assays. In one embodiment, the label is colloidalgold.

Various body fluids are suitable for use in the lateral flowimmunoassays of the instant invention. See, for example, FIG. 19 for anon-limiting list of suitable body fluids. In one embodiment, the bodyfluid is selected from the group consisting of whole blood, serum,plasma, urine, tears, saliva, wound exudate, bronchoalveolar lavagefluid, and cerebrospinal fluid. In a specific embodiment, the body fluidis whole blood.

One skilled in the art will appreciate that various control analytes aresuitable for use in the lateral flow immunoassays of the instantinvention to provide verification that the assay was successfullycompleted. In one embodiment, the control analyte is IgG.

In another embodiment, complement in the body fluid sample is notsubstantially activated experimentally by the lateral flow immunoassayitself.

In one embodiment, the first detecting antibody binds a first epitope ofintact C3, wherein the first epitope of intact C3 is a C3a domain whichis present on intact C3 and which is lost upon activation of C3.

In another embodiment, the first capturing antibody binds a secondepitope of intact C3, wherein the second epitope is a region in the C3ddomain which is present on intact C3, C3b, iC3b, and C3d.

In another embodiment, the second detecting antibody binds a firstepitope of iC3b, wherein the first epitope of iC3b is a neoepitope oniC3b which is revealed when C3b is deactivated to iC3b and which isoccluded when iC3b is further degraded to C3c and C3d.

In still another embodiment, the second capturing antibody binds asecond epitope of iC3b, wherein the second epitope of iC3b is aneoepitope present only on C3b, iC3b, and C3dg.

In another embodiment, the antibodies that bind intact C3 and theantibodies that bind iC3b are not substantially cross-reactive.

EXAMPLES

The following examples are given by way of illustration and are in noway intended to limit the scope of the present invention.

Example 1 Patient Triage

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intactC3 and iC3b standards. Lateral flow immunoassay cassettes are read withan electronic reader after 20 minutes.

The test is used to gauge injury severity within 15, 30, or 60 minutesof injury. It is most useful for patients who may have suffered injuriesnot obvious by visual inspection. A drop of blood is collected eitherfrom an arterial line (A-line) or finger stick. A 10 ul sample is drawnup using a fixed volume pipet. The sample then mixed with 990 ul ofsample buffer. The blood and sample buffer are mixed. Using a fixedvolume pipet bulb, 100 ul is drawn up and pipetted onto the lateral flowimmunoassay cassette containing integrated intact C3 and iC3b teststrips. Alternatively, 100 ul can be applied to separate intact C3 andiC3b lateral flow assay cassettes. After 10 minutes but before 40minutes, the cassette is read and results recorded, preferablyelectronically by a reader. If the first reading has an iC3b level (orequivalent iC3b:intact C3 ratio) higher than 50 μg/ml in blood, evidenceof complement activation and high inflammation exist. Staff assumessevere injury and alerts ER staff. Otherwise, a second reading is taken5 minutes later. If the iC3b level (or equivalent iC3b:intact C3 ratio)is higher than 50 μg/ml in blood or the iC3b level has increased by morethan 25%, the patient is assumed to have severe injury and ER staff isalerted. A lesser increase or no increase is suggestive, but notconclusive, of less severe injury.

Example 2 Trajectory Monitoring of a Trauma Patient

At the beginning of the shift, ICU staff performs a standard curve using10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intact C3 andiC3b standards. Lateral flow immunoassay cassettes are read with anelectronic reader after 20 minutes.

The objective of trajectory monitoring is to detect changes ininflammatory and immune status of patients that have been stabilizedafter severe trauma. In this example, respiratory distress caused byeither pneumonia or inflammatory dysfunction is to be detected. Theexpected patient profile is one who has an injury severity score (ISS)equal or greater to 16 and who requires ventilator assistance forbreathing.

The patient receives a complement test at frequent intervals, whichaligns with the time points for testing glucose levels in blood. Thisinterval between testing is usually about two hours. Blood is collectedusing the same method as for glucose testing, either by A-line or byfinger stick. A 10 ul sample is drawn up using a fixed volume pipet. Thesample then mixed with 990 ul of sample buffer. The blood and samplebuffer are mixed. Using a fixed volume pipet bulb, 100 ul is drawn upand pipetted onto the LFA cassette containing integrated intact C3 andiC3b lateral flow immunoassay cassettes. Alternatively, 100 ul can beapplied to separate intact C3 and iC3b cassettes. The cassette orcassettes will be placed in reader at the patient's bedside. The readeris set to take a reading after 20 minutes. Data is collected and iC3b,intact C3 and iC3b:(intact C3) values recorded at each time point.

Changes in intact C3 or iC3b levels over time or changes in the rate ofchange may indicate a change in inflammatory status. A sharp rise iniC3b, accompanied by a decrease in intact C3, indicates imminentrespiratory distress. As a next course of action, a clinician performs abroncheoalveolar lavage (BAL) on the patient to determine whether thepatient is experiencing VAP. If bacteria are present at levels of 10⁴per ml or higher, VAP is indicate and the patient is places onantibiotic therapy. Otherwise, noninfectious inflammatory dysfunction isassumed and the patient may be treated with anti-inflammatory agentsand/or complement inhibitors. The patient may also have his ventilatorsetting adjusted.

Example 3 Determining Disease Severity and Effectiveness of Treatment ina Patient with Systemic Lupus Erythematosus (SLE)

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml of intactC3 and iC3b standards. Lateral flow immunoassay cassettes are read withan electronic reader after 20 minutes.

The test is used to gauge the initial severity of disease as well theeffectiveness of therapy. One of the standard diagnostics performed onSLE patients is measurement of total C3 levels. C3 levels are normallydepressed in SLE patients and return to normal (>1 mg/ml) followingsuccessful treatment. However, it not known generally whether the C3activation has been abrogated or only slowed enough to allow normalreplenishment mechanisms to restore C3 levels to normal.

At each doctor visit, a patient's blood is collected for total C3,intact C3, and iC3b tests. Only one drop is required for the combine 3tests. Blood is collected by fingerstick unless blood is being drawn forother tests, in which case, the blood will come from that source. A 10ul sample is drawn up using a fixed volume pipet. The sample then mixedwith 990 ul of sample buffer. The blood and sample buffer are mixed.Using a fixed volume pipet bulb, 100 ul is drawn up and pipetted ontothe LFA cassette containing integrated lateral flow cassette thatmeasures total C3, intact C3 and iC3b. Alternatively, 100 ul can beapplied to separate cassettes for each assay. The cassette or cassettesare placed in reader at the doctor's office. The reader is set to take areading after 20 minutes.

Data is collected at each doctor visit. At the initial visit, adding theiC3b and intact C3 tests provides the specialist with more informationabout the severity of the patient's condition than is now possible. Newinformation becomes available at the time that the specialist wouldconsider the patient's status stable. At this point, the iC3b and intactlevels indicate the extent of remaining disease process. If iC3b levels,in particular, are above normal (generally >1%), the underlying diseaseprocess is still very active and the specialist may opt to furtheradjust therapy by increasing anti-inflammatory drug doses or addingadditional medication.

Example 4 Determination of Basal Intact C3 and iC3b Levels in the BasalTear Fluid of a Healthy Individual Over a 24 Hour Period

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, 1000 ng/ml, and 3000ng/ml of intact C3 and iC3b standards. Lateral flow immunoassaycassettes are read with an electronic reader after 20 minutes.

For determining intact C3 and iC3b levels in the eye of a healthyindividuals, three readings in total were taken. Samples were collectedand evaluated of Time=0 hours, 12 hours, and 24 hours.

For tear collection, the lower eyelid is pulled back and briefly dappedwith a Kimwipe® to the lower part of the eye. The Kimwipe® is thenquickly cut where the tear was collected leaving a few millimeters ofdry edge surrounding the tear spot. The Kimwipe®-tear sample is thenplaced into 220 ul of BioAssay Works Diluent Buffer and vortexedthoroughly for 10 seconds. After a one minute wait period, the sample isvortexed again briefly. Next, 100 ul of sample is transferred to eachlateral flow immunoassay (intact C3 and iC3b) and assayed.

For analysis, each cassette is inserted into the reader and read after20 minutes.

The results ranged between 50-60 μ/ml of intact C3 and between 5-8 μ/mlof iC3b (See FIG. 15).

Example 5 Determination of Basal Intact C3 and iC3b Levels in Two HealthIndividuals at a Single Time Point

Before the first test sample is assayed, a standard curve is performedusing 10 ng/ml 30 ng/ml, 100 ng/ml, 300 ng/ml, 1000 ng/ml, and 3000ng/ml of intact C3 and iC3b standards. Lateral flow immunoassaycassettes are read with an electronic reader after 20 minutes.

Resting levels of intact C3 and iC3b are collected from two healthydonors. The lateral flow immunoassay reader is turned on. Finger iscleaned using an alcohol swab. Finger is stuck with lancet and squeezedgently to collect 10 ul of blood using the MICROSAFE® Tube by capillaryaction. Blood sample was expelled directly into a tube filled with 990ml of sample assay buffer and then capped and mixed by inversion 6-8times. 100 ul of blood sample mixture was transferred to CompAct intactC3 test using the 100 ul Exact Volume Pipet. A second 100 ul of bloodsample mixture was then transferred to the CompAct iC3b test using afresh 100 ul Exact Volume Pipet. The timer was set to read after 20minutes for both tests.

The results from the first patient were determined to be approximately500 μg/ml for intact C3 and 200 ng/ml for iC3b. This indicates there is2500 ratio of intact C3 to iC3b in this individual (see FIG. 16). Thesecond individual's results were approximately 1000 μg/ml for intact C3and 300 ng/ml for iC3b (see FIG. 17). Both of these values are withinthe expected normal ranges. The iC3b values are in the lower range ofwhat is considered normal.

Example 6 Determination of Basal Intact C3 and iC3b Levels in a HealthyIndividual at a Single Time Point after Strenuous Exercise

Using the above protocol of Example 5, one of the healthy individualswas tested again after strenuous exercise (see FIG. 18). Exertion didnot significantly alter iC3b or C3 levels.

Example 7 Crosstalk Between Intact C3 and iC3b Antibodies in LateralFlow Immunoassays

In a 1 milliliter volume, 50 ng/ml of iC3b was mixed with varying mountsof intact C3 (ranging from 0 ng/ml to 100,000 ng/ml). Samples were mixedby inversion 6-8 times and then 0.1 ml was pipetted onto the cassette.Readings were taken at 20 minutes. Reader output was converted to iC3bconcentration using a standard curve generated from 10 ng/ml to 100,000ng/ml. Background from a cassette run only with buffer was subtracted.Fractional contributions were calculated by subtracting the actual iC3bconcentration (from iC3b test with no added C3) from apparentconcentration of iC3b at each point and then normalizing against actualiC3b concentrations. See FIG. 14. For the H08K-01 cassettes, at thehighest concentration of C3 tested, about half the iC3b signal came fromintact C3 and half from actual iC3b. For the J24K-03 version, about fourtimes as much iC3b signal came from intact C3 cross talk than fromactual iC3b. Although these antibody pairs work well in ELISA assays,they exhibit significant crosstalk when used in lateral flowimmunoassays for the same analytes. At the physiologically relevant250:1 and 500:1 ratios, intact C3 contributes more to iC3b signal output than iC3b itself in J24K-03.

J24K-03 is an assay with mouse anti-C3a monoclonal on the gold conjugateand mouse anti-C3d monoclonal on the test line. H08K-01 has mouseanti-iC3b monoclonal on the gold conjugate and Anti-C3 polyclonal on thetest line.

Example 8 Generation of iC3b Standard Curve for Lateral FlowImmunoassays

One embodiment of the invention comprises a lateral flow assay stripwithout the cassette casings. These strips had anti-iC3b monoclonal(Quidel® A209) conjugated to the gold and anti-C3 antibody (MPBiomedical® 55237) conjugated to the strip. Standard curves are shown inFIG. 11(A). The standard curves indicated a linear range of about 10fold and a sensitivity of about 100 ng/ml. Another embodiment of theinvention configures the strips for use in a cassette that allowscontrolled application of the sample to the assay strip. This improvedassay-to-assay reproducibility, although there is still considerabletime dependence on the assay. Standard curve results are shown in FIG.11(B). A third embodiment increases the antibody concentration from 0.5mg/ml to 1 mg/ml applied to on the gold conjugate and removes BSA fromthe absorption buffer. Standard curve results are shown in FIG. 11(C).

Standard curves are generated as described in example 9 below.

Example 9 Generation of iC3b Standard Curve for Lateral FlowImmunoassays

Ten (10) μl of a stock of iC3b (concentration 1 mg/ml) was diluted into990 ul of sample dilution buffer to create 10 ug/ml working stock usinga 2 ml capped tube. Tube was mixed by slowly inverting 10-12 times.Investigator diluted 500 ul of 10 ug/ml stock into 500 ul BAW Buffer tocreate a 5 ug/ml stock in another 2 ml capped tube. Mixing was performedby slowly inverting tube 10-12 times. The 1:1 dilution (500 ul:500 ul)was repeated, as described above, nine more times to create thefollowing working stocks:

10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 313 ng/ml, 156ng/ml, 78 ng/ml, 39 ng/ml, 20 ng/ml, 10 ng/ml, and 0 ng/ml (bufferalone).

Lateral flow immunoassay (LFA) cassettes were prepared by labeling andlaying out in groups of three. For each dilution, investigator pipetted100 ul of first working stock (10 ug/ml for intact C3 and 5 ug/ml foriC3b) into sample port of 1st LFA. For each concentration, investigatorwaited 20 seconds before loading 100 ul of same working stock into the2nd LFA. Cassettes were read after 10, 20, and 30 minutes using BioAssayWorks Reader LFDR 101 (Forsite Diagnostics) using the Test line settingfollowed by the Control line setting, and the data recorded.

After the experiment is completed, data was plotted using GraphPad Prism5 software. The standard curve fits the three-parameter logisticequation: Y=Bottom +(Top−Bottom)/(1+EC50/X). See FIG. 12.

Example 10 Generation of Intact C3 Standard Curve for Lateral FlowImmunoassays

Ten (10) μl of a stock of intact (concentration 1 mg/ml) was dilutedinto 990 ul of sample dilution buffer to create 10 ug/ml working stockusing a 2 ml capped tube. Tube was mixed by slowly inverting 10-12times. Investigator diluted 500 ul of 10 ug/ml stock into 500 ul BAWBuffer to create a 5 ug/ml stock in another 2 ml capped tube. Mixing wasperformed by slowly inverting tube 10-12 times. The 1:1 dilution (500ul:500 ul) was repeated, as described above, nine more times to createthe following working stocks:

10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 313 ng/ml, 156ng/ml, 78 ng/ml, 39 ng/ml, 20 ng/ml, 10 ng/ml, and 0 ng/ml (bufferalone).

LFA cassettes were prepared by labeling and laying out in groups ofthree. For each of dilution, investigator pipetted 100 ul of firstworking stock (10 ug/ml for intact C3 and 5 ug/ml for iC3b) into sampleport of 1st LFA. For each concentration, investigator waited 20 secondsbefore loading 100 ul of same working stock into the 2nd LFA. Cassetteswere read after 10, 20, and 30 minutes using BioAssay Works Reader LFDR101 (Forsite Diagnostics) using the Test line setting followed by theControl line setting, and the data recorded.

After the experiment is completed, data was plotted using GraphPad Prism5 software. The standard curve fits the three-parameter logisticequation: Y=Bottom+(Top−Bottom)/(1+EC50/X). See FIG. 13.

All documents cited are incorporated herein by reference; the citationof any document is not to be construed as an admission that it is priorart with respect to the present invention.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to one skilled in the artthat various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A lateral flow immunoassay for the point-of-caredetection of a marker of complement activation in a body fluid samplecomprising complement proteins, the lateral flow immunoassay comprising:a membrane strip; a detecting antibody that binds a first epitope of themarker; a test line comprising a capturing antibody that binds a secondepitope of the marker; and a control line comprising an antibody thatbinds a control analyte, wherein the marker is iC3b.
 2. The lateral flowimmunoassay of claim 1, wherein the detecting antibody comprises a labelthat provides a signal.
 3. The lateral flow immunoassay of claim 1,wherein the second epitope is a region in the C3d domain which ispresent on iC3b.
 4. The lateral flow immunoassay of claim 1, wherein themarker is iC3b and the first epitope is a neoepitope on iC3b which isrevealed when C3b is deactivated to iC3b and which is occluded when iC3bis further degraded to C3c and C3dg.
 5. The lateral flow immunoassay ofclaim 4, wherein the second epitope is a neoepitope present only on C3b,iC3b, and C3dg.
 6. The lateral flow immunoassay of claim 1, wherein thebody fluid is selected from the group consisting of whole blood, serum,plasma, urine, tears, saliva, wound exudate, bronchoalveolar lavagefluid, and cerebrospinal fluid.
 7. The lateral flow immunoassay of claim1, wherein the control analyte is IgG.
 8. The lateral flow immunoassayof claim 1, wherein complement in the body fluid sample is notsubstantially activated experimentally by the lateral flow immunoassay.9. The lateral flow immunoassay according to claim 1 for thepoint-of-care detection of markers of complement activation in a bodyfluid sample comprising complement proteins, the lateral flowimmunoassay further comprising: a second detecting antibody that binds afirst epitope of a second marker; a second test line comprising a secondcapturing antibody that binds a second epitope of a second marker; andat least one control line comprising an antibody that binds a controlanalyte, wherein the second marker is intact C3.
 10. The lateral flowimmunoassay of claim 9, wherein the first and second detectingantibodies each comprise a label that provides a signal.
 11. The lateralflow immunoassay of claim 9, wherein the body fluid is selected from thegroup consisting of whole blood, serum, plasma, urine, tears, andcerebrospinal fluid.
 12. The lateral flow immunoassay of claim 9,wherein the control analyte is IgG.
 13. The lateral flow immunoassay ofclaim 9, wherein complement in the body fluid sample is notsubstantially activated experimentally by the lateral flow immunoassay.14. The lateral flow immunoassay of claim 9, wherein the first epitopeof intact C3 is a C3a domain which is present on intact C3 and which islost upon activation of C3.
 15. The lateral flow immunoassay of claim 9,wherein the second epitope of intact C3 is a region in the C3d domainwhich is present on intact C3, C3b, iC3b, and C3d.
 16. The lateral flowimmunoassay of claim 9, wherein the first epitope of iC3b is aneoepitope on iC3b which is revealed when C3b is deactivated to iC3b andwhich is occluded when iC3b is further degraded to C3c and C3dg.
 17. Thelateral flow immunoassay of claim 9, wherein the second epitope of iC3bis a neoepitope present only on C3b, iC3b, and C3dg.
 18. The lateralflow immunoassay of claim 9, wherein the antibodies that bind intact C3and the antibodies that bind iC3b are not substantially cross-reactive.19. A method for monitoring an individual who is receiving treatment fora physiological condition and who is suffering from acomplement-associated disorder, the method comprising: (a) obtainingserial samples of a body fluid from the individual; (b) determining acomplement activation level in each of said samples via thepoint-of-care lateral flow immunoassay of claim 1; (c) comparing thecomplement activation levels in the serial samples to detect a change ina complement activation level over time; and (d) modifying treatment forthe individual, based on the correlating of step (c).
 20. A method fortreating an individual at risk for a complement-associated disorder, themethod comprising: (a) obtaining a sample of a body fluid from theindividual; (b) measuring a complement activation level in the samplevia the point-of-care lateral flow immunoassay of claim 1; (c)correlating the complement activation level in the sample to a risk of acomplement-associated disorder by comparing the complement activationlevel in the sample to a reference level in a control, wherein adeviation in complement activation level in the sample compared to thereference level in the control indicates the individual is at risk for acomplement-associated disorder; (d) selecting a treatment for theindividual, based on the correlating of step (c); and (e) treating theindividual with the treatment selected in accordance with step (d). 21.The method of claim 20, wherein the complement-associated disorder isselected from the group consisting of trauma, inflammatory distress,autoimmune disorders, intracranial hemorrhage, infection, transplantrejection, ocular disease, heart disease, ischemia/reperfusion injury,age-related macular degeneration, paroxysmal nocturnal hemoglobinuria(PNH), hereditary angiodema, renal disease, pregnancy-associateddisorders, and neurological disorders.
 22. The method of claim 21,wherein the inflammatory distress is selected from the group consistingof organ failure, systemic inflammatory response syndrome (SIRS), adultrespiratory distress syndrome (ARDS), sepsis, and pneumonia.
 23. Themethod of claim 20, wherein the body fluid is selected from the groupconsisting of whole blood, serum, plasma, urine, tears, saliva, woundexudate, bronchoalveolar lavage fluid, and cerebrospinal fluid.
 24. Themethod of claim 20, wherein the lateral flow immunoassay detects thepresence or absence of one or more of intact C3 and iC3b in the sample.25. The method of claim 20, wherein the treatment comprises performingadditional testing on the individual, optimizing a ventilator, oradministering a therapeutic agent selected from the group consisting ofantibiotics, anti-inflammatory agents, and inhibitors of complement. 26.The method of claim 25, wherein the additional testing comprisesobtaining a broncheoalveolar lavage (BAL) sample from the individual.27. The method of claim 25 wherein the inhibitor of complement isselected from the group consisting of natural complement inhibitors andderivatives thereof, compstatin and analogs thereof, anti-membraneattack complex (MAC) antibodies, anti-C3 antibodies, anti-05 antibodies,C3a receptor antagonists, and C5a receptor antagonists.
 28. The methodof claim 20, wherein the point-of-care lateral flow immunoassay providesa measurement of the amount of iC3b and further comprises measurement ofthe amount of intact C3 and/or total C3 in the sample in about 30minutes or less.